Scanning Microscopy Scanning Microscopy

Volume 5 Number 3 Article 14

8-27-1991

Heterogeneity of Crystals Attached to the Human Enamel and Heterogeneity of Crystals Attached to the Human Enamel and

Cementum Surfaces After Calculus Removal In Vitro Cementum Surfaces After Calculus Removal In Vitro

T. Kodaka Showa University

K. Debari Showa University

M. Yamada Showa University

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Recommended Citation Recommended Citation Kodaka, T.; Debari, K.; and Yamada, M. (1991) "Heterogeneity of Crystals Attached to the Human Enamel and Cementum Surfaces After Calculus Removal In Vitro," Scanning Microscopy: Vol. 5 : No. 3 , Article 14. Available at: https://digitalcommons.usu.edu/microscopy/vol5/iss3/14

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Scanning Microscopy, Vol. 5, No . 3, 1991 (Pages 713-721) 0891 -7035/91 $3. 00 + .00 Scanni ng Microscopy Intern ational, Chicago (AMF O'Hare), IL 60666 USA

HETEROGENEITY OF CRYSTALS ATTACHED TO THE HUMAN ENAMEL AND

CEMENTUM SURFACES AFTER CALCULUS REMOVAL IN VITRO

l* 2 1 T. Kodaka , K. Debari and M. Yamada

1 second and 2First Departments of Oral Anatomy, School of Dentistry, Showa University, Tokyo 142, J apan

(Received for publication July 19, 1991, and in revised form August 27, 1991)

Abstract

Twenty one extracted human teeth with dental calc uli on the ena me l and cementum surfaces , fixed in 10% neutral for ma 1 dehyde, were se 1 ected for this study . After ethano l dehydration and air drying, these calculi were removed by tweezers to observe the teeth surfaces under them. The inspection of these surfaces using SEM and EDX revea 1 ed hexahedrally based crysta ls including pseudocuboida 1 , rhombohedra 1 and var iab 1 e rugged rocky shapes. These crystals were identified as Mg­co ntaining whitlockite. The pseudocuboidal crystals , measuring about 4.5 µmin maximum l ength , were widely distributed on the cervica l ename l surface previously covered by calculus. On the root surface, however, these areas decreased remakably; the shapes c hanged from pseudocubes into rhombohedrons and rugged rocky structures, while their sizes were smaller and the Mg content decreased. The difference in frequency and morphological variation of the hexahedrally based crystals might be ca used by the different characteristics of enamel and cementum surfaces and the Mg present on these surfaces.

KEY WORDS. human dental ca l c ulus, mechanical removal, e nam e l surface, cementum surface , SEM, SEM-EDX, hexahedra l ly based crysta ls, distrib u t ion, morp hology, Mg- co nt aini ng whitl ockite

*Address for correspondence : Second Department of Ora l Anatomy, Show a University, School of Den ti st ry, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142, Ja pan. Phone:03-3784-8157

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Introduction

Organic components inc 1 uding ora l microorganisms and the mineral phase of the calculus-tooth interface have been reported by a number of studies [Zander, 1953; Mand e l et al . , 1957; Turesky et al., 1961; Schroeder , 1969; Selvig, 1970; Jones, 1972; Canis et al.,1979; Eide et al. , 1983; Busscher et al., 1989]. It has been demonstrated that Mg­containing whitlockite is a major constituent of dental calc ulus [Schroeder and Bambauer, 1966; Jensen and Rowles, 1967; Gr¢n et al., 1967; Kani et al., 1983], often existed in the innermost calculus layer adjacent to the tooth surface [Kodaka et al., 1988, 1989; Kodaka and Miak e , 1991]. I n addition, the calcul us whitlockite showed various shapes and sizes although they were based on hexahedrons [ Kodaka et al., 1988].

According to Everett and Potter [1 959], human subgingival calculus was divided into ledge or ling-like formation (59%), finger and fern -l ike formations (19%), spiny or nodular deposits (62%), and others . In this study the teeth surfaces , a f ter the removal of the dental calcu l i, were observed by scanning electron microscopy (SEM). The Mg content was detected by e n ergy dispersive e l ectron probe microanalysis (EDX).

Materials and Methods

Twenty- one human t eeth wit h typical forms of dental calcu li (l edge -like, finger -lik e, and spiny ) [ Everett and Potter, 1959], were selected from e xtra cted te et h. These teeth were fixed in 10 % neutral form a ldehyd e for abo ut o ne week. They were c lassifi ed into f ive types based on the position of the ca l culus deposits ( see Fig. 1: A to E). As shown in the low er right of Figure 1 (F), their positions we r e divided into ename l surface (ES), the cementum - enamel

T. Kodaka et a 1.

junction (CEJ) area or the cervical-root surface adjacent to the CEJ (CS-1), and the cervical-root surface away from the CEJ (CS-2). Figure 1 shows that A, B, and C types are ledge-like deposits attached on 1 y to the ES (A type, one sample), from the ES to the CS-1 (B type, four samples), and only in the CS-2 (C type, five samples); the D type is finger-like formed deposits attached to the ES , CS-1, and CS-2 (four samples); and E type is spiny deposits attached to the CS-2 only (seven samples).

The teeth regions without calculus were cut off horizontally and vertically with a diamond wheel. The remaining parts were rinsed in running tap-water, dehydrated with ethanol, dried in the air, and coated with carbon in a Hitachi HUS-5GB high vacuum evaporator. After a 11 the samp 1 es had been set up under a high vacuum in the evaporator, the calculus could be easily removed from the teeth surfaces with tweezers.

After calculus removal, thenon­carbon coated surfaces clearly appeared. These surfaces previously covered by calculus were distinguished macroscopically. The outlines were rough 1 y drawn in Figure 1. The teeth surfaces observed in th e SEM came from nine samp 1 es in the ES (A, B , and D types), eight samples in the CS-1 (B and D types), and 16 samples in the CS-2 (C, D, and E types). Al 1 of them were observed by a Hitachi S-430 SEM after coating with a 10 to 15 nm thick platinum-palladium layer using an Eiko ion sputtering apparatus. The crystals on the teeth surfac e s and the t e eth surfaces were qualitatively analyzed by a Hitachi X560 SEM fitted with a Kevex 7000Q EDX under conditions of 1_5t0kv accelerating voltage and 1 x 10 A specimen irradiation current. In addition, magnesium (Mg) content of larger masses of crystals was quantitatively analyzed by the SEM-EDX under the s.ame conditions. The standard sample for Mg was magnesium oxide and ZAF corrections were used [Moll, 1977].

Results

Figures 2 to 19 are the SEM micrographs of the teeth surfaces after mechanica 1 remova 1 of den ta 1 ca 1 cu 1 us. Figures 2 to 7 show the cervical enamel surfaces (ES; A, B, and D types from Fig. 1). Hexahedrally based crystals were always found in the A and B types of ledge-like deposits. The areas with attached crystals covering over 1/3 of the whole surface were observed in three samp 1 es of A and B types, which had dental calculus attached. Their frequency was "+ 3" (Table l ; Figs. 2 and 4). In the two remaining B type samples

714

/ ~ / '_,- ( ~ ( ') '1 I

I I

l

t

\ \

\

\ A C

~ ..,.,-~ '

~ I ES

Fig. 1. Classification of teeth with attach e d dental calculi (A to E types). Lower-right illustration (F) shows the enamel surface (ES), the CEJ area or the cervical - root surface adjacent to the CEJ (CS-1), and the cervical-root surface away from the CEJ (CS-2). A to C types: ledge-like deposits; A type on the ES, B type on the ES and CS-1, and C type on the CS-2. D type: finger-like form e d deposits on the ES, CS-1, and CS-2. E type: spiny deposits on the CS-2.

scattered small areas with crystals were found with "+2" frequency (Table 1). In the D type one samp 1 e showed the frequency of "+2", but the remaining three samples showed only a few smal l areas of crystals. The frequency was "+l" (Table 1).

The hexahedrally based crystals on the cervical enamel surface showed a pseudocuboidal shape (Figs. 2, 3, 6, and 7) and groupings of quadrang 1 ar b 1 ades cubical ly arranged or variable rugged rocky shapes (Figs. 4 to 6). Al though many of them were more or less fused to each other their sizes measured about 0.2 to 4.5 µmin length. The cervical enamel surface except for the attached crystals showed areas of fine granular and smooth surface structures (Figs. 3, 6, and 7). On some areas, the square windows surrounded by the granular structures were observed (Figs. 6 and 7).

Hexahedral crystals attached to teeth surfac es

The distribution of areas with attached hexahedrally based crystals remarkably decreased on the cervica l­root surface ( lower of Fig. 4). Figures 8 to 13 show the root surfaces after calculus removal (CS-1; Band D types in Fig. 1). Slight elevations dome-shaped of various sizes formed the cementum surface (Fig. 11). As shown in Table 1, the distribution of these areas had the frequency of "+2" in one sample of the B type group (Figs. 8 to 10), but the remaining three samples and one sample of the D type showed the frequency of "+1" (Figs. 11 to 13). In the remaining three samples of the D type, hexahedrally based crystals were rarely found. Their frequency was "±" (Table 1).

The hexahedrally based crysta ls on the root surface showed pseudocuboidal (Figs. 8 to 10) and rhombohedral shapes

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Figs. 2 to 7. Hexahedrally based crystals on the enamel surface [ES]. The c rystals basically show a pseudocuboidal shape. Th e maximum size measur e s about 4.5 µmin length (Fig. 7). Arrowhead in Figure 4: the CEJ. Arrows in Figures 6 and 7: square windows of a smooth surface surrounded by fine granu 1 ar structures.

(Figs. 11 to 13). Some rhombohedral crystals fused to each other and formed rugged rocky structures (Figs. 12 and 13). Their sizes measured about 0.2 to 3 µmin length (Fig. 13). The root surface except for the attached crystals was covered with microorganiSIT)S (Fi g. 10) and fine needle-shaped or granu lar structures (Figs. 10 to 13), and the square windows surrounded by the needle­shaped structures were observed on some areas (Fig. 13).

T. Kodaka et al.

Figures 14 to 19 show the cervical­root surface away from the CEJ after calculus removal (CS-2; C, D, and E types in Fig. 1). Dome-shaped elevations forming the cementum surface were considerably flattened (Figs. 16 and 18). As in Table 1, the distribution areas of hexahedrally based crystals showed the frequency of "+2" in two samples of the C type and the E type, and the frequency of "+l" was seen in the remaining nine samples of the C and E types, but a 11 samp 1 es in the D type showed the rare frequency of "±".

On the root surface, there were masses of fine rhombohedral crystals (Figs. 14 to 16), aggregations of fine pseudocuboidal crystals (Fig . 17), and rhombohedral crystals with the masses fused to each other (Figs. 14 , 18, and 19). These masses and aggregations had variable rugged rocky shapes. The sizes

716

Fi gs . 8 t o 13. Hexahedrally based crysta 1 s on the CEJ area [ CS-1 J. The crystals basically show pseudocuboidal and rhombohedra 1 shapes, a 1 though some of them fuse to each other and form rugged rocky structures (Figs. 12 and 13). The maximum size measures about 3 µmin length (Fig. 13). Microorganisms (Fig. 10) and fine needle-shaped structures are seen (Figs. 10 to 14). Arrows in Figure. 12: square windows surrounded by granular structures.

measured about 0.1 to 2.5 µmin length , although some masses were l arger (Fig . 15). On th .e cementum surface except for attached the crystals and microorganisms (Fig. 17), poor granular structures were observed (Figs. 16 and 1 7). The faint square windows surrounded by the granular structures could be seen on some areas (Fig. 16).

Hexahedral crystals attached to teeth surfaces

During the analysis by the SEM-EDX, the Mg element was not detected on the smooth and fine needle-shaped and granular structures on the ena mel and cementum surfaces, but always detected from the hexahedrally based crysta ls. Table 2 displays the Mg concent rations detected on 1 arger masses of the crystals in six samples: Al, Bl, B2, Cl , Dl, and El (* in Table 1). The mean Mg content was higher in the crystals on the enamel surface (ES) than those on the cementum surfaces (CS-1, CS-2), but there was no significant difference (E < 0.05) between them.

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Figs. 14 to 19. Hexahedra l ly based crystals in the cervical -root surface [CS-2]. Most of the crystals show fine and sma 11 rhombohedra 1 shapes and some of them a fine pseudocuboidal shape, although many crystals form variable rugged rocky structures. Th e maximum size measures about 2.5 µm in length (Fig. 18). Microorganisms and poor granular structures are also seen in Figure 17. Arrows in Figure 16: square windows surrounded by the granular structures.

T. Kodaka et al.

Table 1. Frequency of hexahedrally based crystals attached to the tooth surface after calculus removal.

---------------------------------------Calculus position

Type Sample No.

Al

ES

+3*

CS-1 CS-2

--------------------------------------Bl B2 B3 B4

+3 +3* +3 +2

+2* +1 +1 +1

--------------------------------------Cl C2 C3 C4 cs

+2* +1 +1 +1 +1

--------------------------------------D1 +2 +1* ± D2 +1 ± ± D3 +1 ± ± D4 +1 ± ±

El +2* E2 +1 E3 +1 E4 +1 ES +1 E6 +1 E7 ±

--------------------------------------Mean

(~)

+2.1 (9)

+0.8 (8)

+0.8 ( 16)

ES: enamel surface. CS-1: CEJ area. CS-2: cervical-root surface. +3: wide areas over 1/3. +2: small areas scattered here and there. +1: a few small area . ±: rare. A to E type: see Fig. 1. *: quantitatively analyzed by the SEM­EDX (see Table 2).

Table 2. hexahedrally analysis.

Mg based

concentration of crystals. SEM-EDX

Calculus position

Mg content (weight%)

ES CS-1 CS-2

0.72±0 .49 0.50±0.42 0.54±0.32

[ 0.22 - 1.51) [0.08 - 1.13) [0.09 - 1.34]

Mean±S.D. (~=10) [Min imum-Maximum)

ES: enamel surface. CS-1: CEJ area. cs-2: cervical-root surface.

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Discussion

Hexahedral ly based crystals observed in human dental calculus are either in number of whitlockite or brushite crystals [N ewese ly, 1965; Schroeder and Bambauer, 1966; Schroeder, 1969; Dr.iessens, 198 2 ; Moriwaki et al., 1983). From the detection of a small amount of Mg (Table 2), the hexahedrally based crystals including pseudocuboidal, rhombohedral, and variable rugged rocky shapes (Figs. 2 to 19), observed in this study, were identified as Mg-containing whitlockite [J ensen and Rowles, 1967; Santos and GonzAlez-Dlaz, 1980; Kodaka et al., 1988, 1989; LeGeros et al., 1988, 1989).

According to our previous studies [Kodaka et al., 1988, 1989; Kodaka and Miake, 1991), the whitlockite crystals of supragingival calculus were frequently seen in the innermost layer and in the intra-spaces of the deposits. However, the crysta 1 s were not found in the outer surface exposed to the oral cavity. Th e crystals covered the outer surface layer exposed to gingival fluid in marginal (supra- and subgingival ) ledge-like and subgingival spiny deposits , a 1 though the ca lcu 1 us surface facing to the oral cavity in the margina 1 ledge-1 ike deposits were similar to supragingival calculus. The present SEM resu 1 ts of the observation of teeth surfaces after calculus removal were consistent with the above-mentioned data.

Mg-containing whitlockite crystals are formed by higher Mg sources and a rise of pH of saliva and gingival fluid than other dental crystals [Newesely, 1965; Gr(J!n et al., 1967; Killian and Ennever, 19 7 5; Boyan-Sa 1 yers et a 1., 1978; Knuuttia et al., 1980; Driessens, 1982; Kani et al., 1983; LeGeros et al., 1988, 1989). According to our previous study [K odaka and Miake, 1991) (see Table 3) , the Mg content was smaller in the inner than in the outer layer of subgingival spiny deposits attached to the root surface, whereas the Mg content increased from the outer to the inner layer of marginal ledge-like deposits attached from the cervical enamel to the root surface. There was no significant difference in Mg content in these inner layers. The SEM-ECX ana lysis (Table 2 ) showed no signifi ~ant difference of Mg content in the hexahedra lly based crystals attached Lo the teeth surfaces, and the Mg element was not detected on the teeth surfaces, only on the crystals.

The frequency of the hexahedrally based crystals (Table 1) and the Mg content on the tooth surface (Table 2) indicate that the supplied Mg was higher

Hexahedral crystals attac hed to teeth surfaces

on the cervical enamel than on the root surface ; and a s mal 1 increase of pH mi gh t be seen on the in terfa ce of the ca l c ulu s and the ena me l surface. Th e freq uen cy of the crystals may be ca us ed by the difference of the supplied Mg content on the teeth surfaces ; in addition , the morphological variation as shape and size might be a lso influenced by the diff e r e nc e of the supplied Mg content and pH range. The Mg source may be derived from saliva, or gingival fluid as wel l which can penetrate the calculus -t ooth interface [Jenk ins , 1978; Dr iessens , 19 8 2 J. The ename 1 minera 1 with attached deposits might promote th e Mg-containing crysta ls forma tion as shown in enamel caries [H elmcke , 1955; Newesely and H~hling, 1964; Lester and Boyde, 1968] or arrested enamel caries [Plackova and Vahl , 1967], as well as in a case of arrested dentin caries [Daculsi et al., 1979, 1987; LeGeros, et al. , 1989].

Fine need 1 e - shaped and granu 1 ar structures observed on the teeth surfaces of air - dried samples is not organic matter but mineral (Figs. 3 , 6 , 7 , 10, 12, 13, and 17), and is probably some kind of apatite [Lu stmann et al ., 1976; Kodaka et al., 1988]. The shape and density of the fine structures were poor and 1 ower on the cementum when c ompared to the enamel as well as the hexahedra 11 y based crysta 1 s , whereas oral microo .rganisms [K odaka et a l., 1989] tended to be observed only on the c ementum surface (Figs. 11 and 18).

The square windows surrounded by the fine ne edle and granular structures

Table 3. Mg concentration of ledge-like deposits (A and B types ) attached from the enamel sur fa ce (ES ) to the CEJ area (CS-1) and spiny deposits (E type) attac h ed to the cervical-root s urfac e (CS-2).

Ca lculus

Outer lay er * Middle lay er * Inn er layer

Mg content (weight%)

Ledge -like deposits

(A, B types)

0.60±0.91 0.96±1.03 1. 04±1. 21

Spiny deposits (E types)

2 .40± 0 . 82 2 .10±0. 86 1.27±1.09

Mean ±S.D. (.Q_=25)

*: significance l eve l All dat a were cited Miake [1991]

(.e < 0. 01) from Kodaka and

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had traces of hexahedra l ly based crystals (Figs. 6, 7, 1 2 , and 16). These c rystals probably were remov ed with the calculus when the physical force was applied or removed by the vacuum of the eva porat or chamber. The square surfaces were covered with a ve r y thin organic membrane, i.e., pelli cle as prev iously reported, if it exists . It is, therefore, suggested that there are sma ll er amounts of o rgani c substances on the in terface between the deposit and the ename 1 surface (A and B types in Fig. 1) than that of the d eep subgingival dep osits and the root surface (C and E types in Fig. 1). In other words, the inner ca 1 cu 1 us surf ace adjacent to the ename l surface has been fully min era lized as compared wit h that adjacent to the ceme ntum surface.

The finger-like for med deposits distrib ut ed from the enamel to the ce mentum s urface (D type in Fig. 1) were a 1 so observed . As shown in Tab 1 e 1, the ledge-like and spiny deposits (A, B, C, and E types) showed frequent hexahedral l y based crystals simi l ar to eac h other in the ename l and the root surface. The D type of the finger-like formed deposits, however, was somew hat different from the others . Th ey showed less hexahedrally based crystals than the A and B types in the ena me 1, and also l ess of the same crysta l s than the B, C, and E types in the root surface . Though this appearance in the finger ­like formed deposits might be caused by their morphological characteristic and formation , it could not be elucidated in this study.

Considering the clinical app 1 ica tions of these findings, t h e hexahedrally based crysta ls, Mg­containing whit 1 ocki te , may remain when the l edge-like deposits attached to the teeth surfaces are removed with a scaler . The re maining crysta l s strongly attached to the cervical enamel surface could facilitate plaque attachment, and s ubs eque nt mineraliz ation could begin there.

For References and Discussion with Reviewers, seep 720-7 21.

T. Kodaka et al.

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in human subgingival calculus. Scana . J. Dent. Res. 88: 513-516.

Kodaka-T, Debari K, Higashi S (1988). Magnesium-containing crystals in human dental calculus. J. Electron Microsc. 37: 73-80.

Kodaka T, Hirayama A, Miake K, Higashi S (1989). Bacillus-shaped deposits composed of hexahedrally based c rystals in human dental calculus. Scanning Microsc. 3: 843-854.

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Hexahedral crystals attached to teeth surfaces

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Dis cu ss ion wit h Re v ie wers

G. Daculsi: You described some joined crystals as " fused crystals " and in discussion you suggest a fusion process. What evidence do you have for crystal fusion ? What do you think of an epitaxic growing process? Authors: The fused whitlock ite crysta ls have been reported with SEM in synthetic calcium phosphates [L eGeros et al., 1988], arrested enamel caries [Lester and Boyde, 1968], in renal calculus [Santos and Gonzallez-Diaz, 1980], and in dental calculus [ Kodaka et al . , 1988 , 1989; LeGeros et al., 1988 , 1989] . Some of these crystals might have been formed by the epitaxial growth on the previously formed or biological aptites. Aoba et al. [ 1974] reported the epitaxia l growth of whitlockite on the ename l apatite in vitro. In this study , the higher frequency-of whitlockite on the enamel surface than on the cementum surface might be caused by the high purity of the apatite.

M. Alves : How do you know what is supra - and subgingiva l calcu l us if t h e teeth were extracted? Authours: The c l assification of dental ca lcuTus attached to human teeth by Everett and Potter [ 1959] was followed . They classified ledge - like, finger - like , and spiny deposits as subgingival , but we regarded the ledge-like deposits as the transitional type between supra- and subgingival calculus because the calculus is exposed to both the oral cavity and gingival pocket sides [ Kodaka and Miake , 1991].

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Addit i onal Re fe r ence

Aoba T , Ishida T , Hasegawa K, Moriwaki Y, Yamauchi T (1974) . Epitaxia l growth of brushite and whitlockite on the biological apatite {in Japanese} . Jpn. J . Oral Biol. l:__§_: 252-259.